Materials and Reagents

1. PYREX^® disposable glass conical centrifuge tubes without cap (capacity: 50 ml) (Sigma-Aldrich, catalog number: CLS9950250-72EA )
   
2. Disposable screw thread culture tubes with marking spot (ø13 mm) (KIMAX test tubes with Teflon liner caps) (Thomas Scientific, catalog number: 9210J23 )
   
3. Pasteur pipettes (non-plugged, L 5 ¾ in.) (Sigma-Aldrich, catalog number: S6018 )
   
4. Polycarbonate membranes (Avanti Polar Lipids)
   Note: The diameter of their pores is defined by the manipulator (Example: 100 nm, Avanti Polar Lipids, catalog number: 610005 ).
   
5. Grids for transmission electron microscopy (grid size 400 mesh x 62 μm pitch, copper) (Sigma-Aldrich, catalog number: G5026 )
   
6. 0.80 ml open ultra-clear centrifuge tubes (size 5 x 41 mm) (Beckman Coulter; catalog number: 344090 ) and split adaptors (Beckman Coulter; catalog number: 356860 )
   
7. Nitrocellulose membranes for protein transfer as for example: nitrocellulose membranes, 0.2 µm, 8 x 12 cm (Thermo Fisher Scientific, catalog number: 77012 )
   
8. HeLa cells (ATTC, catalog number: CCL-2 )
   
9. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, catalog number: 41966-029/052 )
   
10. Fetal bovine serum (FBS) (Eurobio, catalog number: CVFSVF0001 )
    
11. Penicillin/streptomycin (PAN-Biotech, catalog number: P06-07100 )
    
12. L-glutamine (200 mM) (Thermo Fisher Scientific, catalog number: 25030-024 )
    
13. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190-094/069 )
    
14. Chloroform (for HPLC, ≥ 99.8%, amylene stabilized) (Sigma-Aldrich, catalog number: 34854 )
    
15. Methanol (for HPLC, ≥ 99.9%) (Sigma-Aldrich, catalog number: 34860 )
    
16. Water sterile-filtered (BioReagent, suitable for cell culture) (Sigma-Aldrich, catalog number: W3500 )
    
17. C21:0 fatty acid (Heneicosanoic acid) (Matreya, catalog number: 1241 )
    
18. Hexane (anhydrous, 95%) (Sigma-Aldrich, catalog number: 296090 )
    
19. Sulfuric acid (99.999%) (Sigma-Aldrich, catalog number: 339741 )
    
20. HEPES free acid [N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid); 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] (Sigma-Aldrich, catalog number: H3375 )
    
21. Sodium chloride (NaCl) (ACS reagent, ≥ 99.0%) (Sigma-Aldrich, catalog number: S9888 )
    
22. Liquid N₂
    
23. Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400 )
    
24. Sucrose (≥ 99.5%) [α-D-Glucopyranosyl, β-D-fructofuranoside, D(+)-Saccharose] (Sigma-Aldrich, catalog number: S8501 )
    
25. Iodine (ACS reagent, ≥ 99.8%, solid) (Sigma-Aldrich, catalog number: 207772 )
    
26. Complete DMEM medium (see Recipes)
    
27. Protein-lipid binding buffer (see Recipes)
    

Equipment

1. Flasks (150 cm²) (Dominique Dutscher, catalog number: 190150 )
   
2. 37 °C/5% CO₂ cell culture incubator (Dominique Dutscher, catalog number: 911378M )
   
3. Refrigerated bench centrifuge (Dominique Dutscher, catalog number: 472456 )
   
4. Fume hood (standard equipment of any lab)
   
5. Vortex (Dominique Dutscher, catalog number: 079030 )
   
6. Bottle of nitrogen gas
   
7. Freezer (-20 °C) (Dominique Dutscher, catalog number: 099288B )
   
8. 10 μl positive displacement pipet such as microman Gilson model M10 (Gilson, catalog number: F148501 )
   
9. Oven (for dry heat: 100 °C) (Dominique Dutscher, catalog number: 780405 )
   
10. BPX70 gas chromatography column (30 m x 0.25 mm ID BPX70 0.25 μm) (Trajan Scientific, catalog number: 0 54622 )
    
11. Gas chromatography apparatus (Shimadzu, ref GC-2010 Plus High-end GC)
    
12. Water bath (37 °C) (Dominique Dutscher, catalog number: 910648 )
    
13. Extruder set with holder/heating block (Avanti Polar Lipids, catalog number: 610000 )
    
14. 2 x 250 μl Hamilton^® GASTIGHT^® syringes, 1800 series (1825N, volume 250 μl, needle size 22s ga [bevel tip], needles L51 mm [2 in.]) (Sigma-Aldrich, catalog number: 21394 )
    
15. Fridge (4 °C) (Dominique Dutscher, catalog number: 670251B )
    
16. Rotating agitator (Dominique Dutscher, catalog number: 062646 )
    
17. Carbon evaporator (Q150T Turbo-Pumped sputter coater/carbon coater) (Quorum Technologies, model: Q150T )
    
18. Electron transmission microscope such as JEOL-1400 plus (MET 120 kV) (JEOL, model: 1400 Plus )
    
19. Ultracentrifuge type TL100 labtop equipped with an MLS-50 Swinging-Bucket rotor (Beckman Coulter, catalog number: 367280 )
    
20. 12% Mini-PROTEAN^® TGX^TM precast protein gels (10-well, 30 µl) (Bio-Rad Laboratories, catalog number: 4561043 )
    
21. Power supplier (Dominique Dutscher, catalog number: 049192 )
    
22. SDS-PAGE apparatus (SDS-PAGE), as for example Mini-PROTEAN^® Tetra vertical electrophoresis cell for mini precast gels, 2-gels (Bio-Rad Laboratories, catalog number: 1658005 )
    
23. Protein transfer apparatus, as for example Trans-Blot^® Turbo^TM transfer system (Bio-Rad Laboratories, catalog number: 1704150 )
    
24. Thin layer chromatography plates: silica gel on TLC aluminium foils (silica gel matrix, L x W = 20 x 20 cm) (Sigma-Aldrich, catalog number: 60805 )
    
25. Hermetic glass tank such as Aldrich^® rectangular TLC developing tanks, complete (L x H x W = 27.0 x 26.5 x 7.0 cm) (Sigma-Aldrich, catalog number: Z126195 )
    

Procedure